Engineering of stable enzymes and robust therapeutic proteins is of central importance to the biotechnology and pharmaceutical industries. The primary internal driving forces for stabilizing proteins involve various interactions such as desolvation, electrostatic interaction, hydrogen bonding, and van der Waal forces, and a proper balance of these interactions is necessary for the appropriate folding of a protein. Although protein engineering provides powerful tools for the enhancement of enzymatic activity and protein stability (J. L. Cleland, C. S. Craik, Protein Engineering: Principles and Practice (Wiley-Liss, New York, N.Y., 1996); D. Mendel, J. A. Ellman, Z. Y. Chang, D. L. Veenstra, P. A. Kollman, Science 256, 1798 1992; A. R. Fersht, L. Serrano, Curr. Opin. Struct. Biol. 3, 75 1993; B. W. Matthews, Adv. Protein Chem. 46, 249 1995), the scope of engineering of proteins is limited by the functionality offered by the twenty naturally occurring proteinogenic amino acids (V. W. Cornish, D. Mendel, P. G. Schultz, Angew. Chem. Int. Ed. Engl. 34, 621, 1995), permitting only modest and unpredictable gains in stability by modifying the protein sequence.
Non-natural amino acids that contain unique side chain functional groups including halogens, unsaturated hydrocarbons, heterocycles, silicon, organometallic units, can offer advantages in improving the stability of the folded structure of proteins without requiring sequence modifications. Functionalities orthogonal to that of the naturally occurring amino acids, including alkenes (van Hest, J. C. M. et al., 1998. FEBS Lett., 428, 68-70), alkynes (van Hest, J. C. M.; Kiick, L. K.; Tirrell, D. A. J. Am. Chem. Soc. 2000, 122, 1282-1288), aryl halides (Sharma, N.; Furter, R.; Kast, P.; Tirrell, D. A. FEBS Lett. 2000, 467, 37-40) and electroactive side chains (Kothakota, S.; Fournier, M. J.; Tirrell, D. A.; Mason, T. L. J. Am. Chem. Soc. 1995, 117, 536-537) have been incorporated into proteins prepared in bacterial cultures. Trifluoromethionine has been inserted into bacteriophage lambda lysozyme in vivo and serves as a unique probe for 19F NMR spectroscopy (Duewel, H.; Daub, E.; Robinson, V.; Honek, J. F. Biochemistry 1997, 36, 3404-3416). Trifluoroleucine was reported more than 30 years ago to support bacterial cell growth and to be incorporated into nascent proteins in the absence of leucine during biosynthesis (Rennert, O. M.; Anker, H. S. Biochemistry 1963, 2, 471). In addition, substitution of amino acids such as serine or alanine that normally comprise the hydrophillic region(s) of the proteins, but are also present, to a lesser degree, in the hydrophobic regions, with the fluoro derivatives is likely to result in stronger inter-helical interaction, thus resulting in improved stability.
Leucine-zipper domains occur commonly in protein assemblies such as eukaryotic transcription factors (O'Shea, E. K, Rutkowski, R., Kim, P. S. Science 1989, 243, 538-542; Lumb, K. J, Kim, P. S. Science 1995, 268, 436-438; Wendt, H., Baici, A., Bosshard, H. R.; J. Am. Chem. Soc. 1994, 116, 6073-6074; Gonzales, L., Brown, R. A., Richardson, D., Alber, T. Nat. Struct. Biol. 1996, 3, 1002-1100; Kenar, K. T., Garcia-Moreno, B., Freire, E. Protein Sci. 1995, 4, 1934-1938; Mohanty, D., Kolinski, A., Skolnick, J. Biophys. J. 1999, 77, 54-69; d'Avignon, D. A., Bretthorst, G. L., Holtzer, M. E., Holtzer, A. Biophys. J. 1999, 76, 2752-2759). Such domains form coiled-coil structures comprising generic heptad repeats designated abcdefg, where the d positions are occupied predominantly by leucine residues. The thermodynamics (Thompson, K. S., Vinson, C. R., Freire, E. Biochemistry 1993, 32, 5491-5496; Krylov, D, Mikhailenko, I., Vinson, C. EMBO J. 1994, 13, 2849-2861), kinetics (Wendt, H., Berger, C., Baici, A., Thomas, R. M., Bosshard, H. R. Biochemistry 1995, 34, 4091-4107; Chao, H., Houston, M. E., Grothe, S., Kay, C. M., O'Connor-McCourt, M., Irvin, R. T., Hodges, R. S. Biochemistry 1996, 35, 12175-12185) and structural features (O'Shea, E. K., Klemm, J. D., Kim, P. S., Alber, T. Science 1991, 254, 539-544; Nautiyal, S., Alber, T., Protein Sci. 1999, 8, 84-90; Harbury, P. B., Zhang, T., Kim, P. S., Alber, T. Science, 1993, 262, 1401-1407) of leucine zipper peptides have been characterized extensively. Studies in which leucine residues at the d positions have been replaced by other naturally occurring aliphatic amino acids have demonstrated that leucine is the most effective amino acid in terms of stabilization of the coiled-coil structure (Moitra, J., Szilak, L., Krylov, D., Vinson, C. Biochemistry 1997, 36, 12567-12573; Hodges, R. S., Zhou, N. E., Kay, C. M., Semchul, P. D. Peptide Research, 1990, 3, 125-137). In fact, leucine is the most abundant amino acid in cellular proteins spanning a wide range of organisms (Creighton, T. E. Proteins Structures and Molecular Properties; W. H. Freeman and Company: New York, 1993). Leucine-enriched hydrophobic cores are important in driving protein folding and determining protein stability in a large number of proteins (Lubienski, M. J., Bycroft, M., Freund, S. M. V., Fersht, A. R. Biochemistry 1994, 33, 8866-8877; Hill, C. P., Osslund, T. D., Eisenberg, D. Proc. Natl. Acad. Sci. USA 1993, 90, 5167-5171).
Previous examples of employing other natural amino acids as an attempt to replace leucine have all resulted in loss in coiled coil stability (Moitra, J.; Szilak, L.; Krylov, D.; Vinson, C. Biochemistry 1997, 36, 12567-12573; Hodges, R. S.; Zhou, N. E.; Kay, C. M.; Semchul, P. D.; Peptide Research, 1990, 3, 125-137). This is largely due to the fact that these substitutions are usually the “large” to “small” type and can result in loss of protein hydrophobic core packing efficiency (Sandberg, W.; Terwilliger, T. Science 1989, 245, 54-57; Baldwin, E.; Xu, J.; Hajiseyedjavadi, O.; Baase, W. A.; Matthews, B. W. J. Mol. Biol. 1996, 259, 542-559; Kono, H.; Nishiyama, M.; Tanokura, M.; Doi, J. Protein Eng. 1998, 11, 47-52). Protein cores are believed to be tightly packed and require a jigsaw puzzle-like arrangement of different residue side chains (Harpaz, Y., Gerstein, M.; Chothia, C. Structure 1994, 2, 641-649; Richards, F. M., Lim, W. A. Q. Rev. Biophys. 1994, 15, 507-523; Levitt, M., Gerstein, M., Huang, E., Subbiah, S., Tsai, J. Annu. Rev. Biochem. 1997, 66, 549-579). Thus any perturbation with amino acids of slight difference in geometry can result in substantial energetic cost.
The present invention provides a unique strategy to systematically target the hydrophobic core region(s) of proteins, wherein naturally occurring hydrophobic amino acids are replaced with hyper-hydrophobic non-natural amino acids, resulting in the creation of novel artificial polypeptides, which are identical to the corresponding natural proteins in their tertiary structure and function, but offer an additional advantage of increased stability relative to the corresponding wild type proteins.